Table Of ContentReference: Biol. Bull. 197: 34X-360. (December 1999)
Spectral Sensitivity of Vision and Bioluminescence in
the Midwater Shrimp Sergestes similis
S. M. LINDSAY1 *, T. M. FRANK2, J. KENT3. J. C. PARTRIDGE3, AND M. I. LATZ1
Marine Biology Research Division, Scripps Institution ofOceanography, Universitv ofCalifornia
San Diego, La Jolla, California: ~ Harbor Branch Oceanographic Institution. Ft. Pierce, Florida;
and ' School ofBiological Sciences. University ofBristol, United Kingdom
Abstract. In the oceanic midwater environment, many Introduction
fish, squid, and shrimp use luminescent countershading to
remain cryptic to silhouette-scanning predators. The mid- In terrestrial, aquatic, and aerial environments, vision is
waterpenaeid shrimp, Sergestessimilis Hansen, responds to central to many predator-prey interactions. Thus, camou-
downward-directed light with a dim bioluminescence that flage (crypsis) is a common method of predator avoidance
dynamically matches the spectral radiance ofoceanic down- used by a wide variety ofinvertebrates and vertebrates. The
welling light at depth. Although the sensory basis of lumi- oceanic midwaterenvironment offers few structural refuges
nescent countershading behavior is visual, the relationship from predation. Visual predators commonly search for prey
between visual and behavioral sensitivity is poorly under- silhouetted against dim downwelling irradiance. Camou-
stood. In this study, visual spectral sensitivity, based on flage strategies adopted by midwater animals include trans-
microspectrophotometry and electrophysiological measure- parency, reflective camouflage, and photophore-mediated
mentsofphotoreceptorresponse, is directly compared tothe ecoxuanmtpelrei,llusmoimneatiaonnim(arlesvieswuecdhbaysMgceFlaaltli-nNoguasi,zo1o9p9l0a)n.ktFoonr
behavioral spectral efficiency of luminescent countershad-
have a refractive index similar to that of seawater, making
ing. Microspectrophotometric measurements on single pho- them optically transparent (Chapman, 1976). Many fish use
atobrseocrebpatnocrse ratev4e9a5lednmonilnytahesibnlgulee-vgirseueanlrpeiggimoennotfwtihtehsppeeack- breyflseicmtuilveatcianmgotuhfelaanggeultoarbldiesntdriwbiutthiotnhoefooptciecaanlicenlviighrton(mDeenn-t
trum. The peak electrophysiological spectral sensitivity of ton. 1970). Finally, animals may produce downward-di-
dark-adapted eyes was centered at about 500 nm. The spec- rected bioluminescence. disrupting or minimizing their sil-
tral efficiency of luminescent countershading showed a houette (Clarke, 1963; Herring. 1982; Young. 1983); this
broad peak from 480 to 520 nm. Both electrophysiological behavior is termed luminescent countershading or counter-
and behavioral dataclosely matched the normalized spectral illumination.
absorptance curve of a rhodopsin with Amax == 495 nm, For luminescent countershading to be effective, the opti-
when rhabdom length and photopigment specific absor- cal properties of the bioluminescence must match those of
bance were considered. The close coupling between visual the optical environment. Laboratory studies have demon-
spectral sensitivity and the spectral efficiency of lumines- strated that the angular distribution, intensity, and spectral
cent countershading attests to the importance of biolumi- emission ofluminescent countershading from many midwa-
nescence as a camouflage strategy in this species. ter animals match those ofoceanic downwelling irradiance
(reviewed by Young, 1983). Undercertainconditions, some
squid modify the spectral emission of their biolumines-
cence, presumably to match diel changes in the spectral
composition of downwelling light (Young et ai, 1980).
Received 15 December 1998; accepted 23 September 1999. The optical properties of the oceanic midwater environ-
*Present address: School of Marine Sciences. University of Maine, ment derive from downwelling light and bioluminescence,
Orono. Ml: ()44(i9-S74l: E-mail: slmdsaytsniaine.edu and the relative importance ofthese components varies with
348
VISION AND BIOLUMINESCENCE IN SERGESTES SIM11.IS 349
time of day, depth, and distance from the source of light. nl., 1983). irradiance (Warner et ai, 1979). and angular
Far-field illumination consists of diffuse, dim downwelling distribution (Latz and Case. 1982) ofdownwelling oceanic
light that decreases exponentially with depth and has an light. In addition, ventrally directed dim glowing is pro-
irradiance spectrum centered on 475 nm (Jerlov, 1968). duced only when a downward-directed light stimulus is
Near-field illumination consists mainly ofbioluminescence, present and is extinguished within seconds of the cessation
manifested as point sources of light with emission spectra ofthe stimulus (Warner et ai, 1979; Latz and Case, 1992).
peaking primarily between 460 and 490 nm. depending on Luminescent countershading in Sergestes siniilis is
species (Herring, 1983; Widder et ai. 1983; Latz et ai. clearly dependent on vision; covering the eyes reversibly
1988). Deep-sea and midwater animals have well-devel- abolishesthe response (Warneretai, 1979). Shrimp thatare
oped eyes with unique adaptations for dealing with the in completely dark adapted are initially unresponsive to light,
situ light conditions they encounter. Specialized features but continued light exposure induces bioluminescence after
include large ortubulareyes (squids and fishes: Bowmaker, a latency of several minutes, reaching maximum intensity
1976; Locket, 1977), yellow-pigmented lenses that increase about 20 min later (Latz and Case, 1992). Once lumines-
the contrast between bioluminescence and downwelling cence is induced, responses exhibit the typical fast kinetics
light (fishes: Muntz, 1976), rod-dominated retinae in fishes ofluminescentcountershading observed in squid and fishes;
for greater light sensitivity (Bowmaker, 1976), and visual luminescence increases within several secondsoflight stim-
pigments with blue-shifted (470-490 nm) absorption max- ulation and reaches maximum intensity in approximately
ima (fishes: Partridge et ai. 1989; Douglas et ai. 1995; 30 s (Latz and Case. 1992). Thereafter, the lackofeye shine
cephalopods: Kito et ai, 1993: crustaceans: references in suggests that the eyes are light adapted when luminescent
Frank and Case. 1988). These adaptations attest to the countershading occurs.
importance of bioluminescence and vision in the deep sea. In the present study, behavioral and physiological ap-
Thus there may be a close link between visual spectral proaches were used to characterize the link between vision
sensitivity and the spectral efficiency of luminescent and luminescent countershading behavior in Sergestes si-
countershading (i.e., behavioral spectral sensitivity) if milis. Results demonstrate a close coupling between visual
luminescent countershading is to be a successful means spectral sensitivity and the spectral efficiency of lumines-
of camouflage. In the simplest case, the visual system, cent countershading, further supporting the hypothesized
bioluminescence emission, and oceanic downwelling role of bioluminescence in camouflaging this species.
light will all operate in the same spectral range. This
hypothesis has never been tested, largely due to the Materials and Methods
difficulty in obtaining quantifiable behavioral data from Collection ofanimals and tissue
midwater and deep-sea animals. Typically, these organ-
isms survive for very short periods when brought to the Adult specimens of Sergestes siniilis Hansen were col-
surface, and controlled experimental studies of meaning- lected at night from depths of55 to 300 m in the San Diego
ful behaviors have been difficult to achieve. Studies by Trough (8 Dec 1996. 12 Jun 1997. and 25 Jun 1997), the
Land (1992) and Frank and Widder (1994a.b) are the San Clemente Basin (10 Aug 1997). both near San Diego,
only investigations of visually mediated swimming re- California, USA, and in the Santa Barbara Basin (28 Sep
sponses by midwater and deep-sea crustaceans under 1997 and 24 Oct 1997), near Santa Barbara. California,
environmentally relevant illumination levels. The only during cruises of the RV R.G. Spnnil. Animals were col-
examination ofthe spectral efficiency ofbioluminescence lected using a modified Tucker trawl with a closing light-
in a marine organism was performed by Kay ( 1965) with proofcodend(afterChildressetai, 1977). Thecodendwas
the euphausiid crustacean, Meganyctiphan.es norvegica, closed at depth and animals were brought to the surface and
although the intensity of the photoflash stimulus em- sorted in 5C seawater under dim red light. Animals were
ployed was considerably brighter than in situ levels ex- placed in light-proof containers filled with chilled seawater
perienced by the animal. and transported to the shore laboratory, where they were
The present study investigates visual and behavioral sen- maintained in constant darkness in aquaria with flow-
sitivity in the bioluminescent penaeid shrimp, Sergestes through, 5 /nm filtered seawater at a temperature of 10C.
siniilis. a common member of the midwater community in All experiments were performed within one week of col-
the northeast Pacific Ocean. Bioluminescence in S. siniilis lection; only actively swimming specimens were used for
originates from modified portions of the hepatopancreas, testing. Animals were not fed. and except forbriefexposure
called organs of Pesta, which produce ventrally directed to dim red light during handling, they remained in constant
light. Previous laboratory studies indicate that the emitted darkness.
light is consistent with a camouflage function. Biolumines- Forelectroretinogram studies, animals collected on 8 Dec
cence is tuned to the optical properties of the midwater 1996 from the San DiegoTrough were shipped in light-tight
environment, matching the spectral distribution (Widder et containers to the Harbor Branch Oceanographic Institution
350 S. M. LINDSAY ET AL.
(HBOI), Fort Pierce, Florida. At HBOI. shrimp were main- 1996) were fitted to the measured difference spectra to
tained in chilled (10C) filtered seawater in constant dark- provide estimates of the Amav of R and M pigments (Par-
ness. tridge and De Grip, 1991; Hart etaI., 1998). The fraction of
Formicrospectrophotometry experiments, adult S. siniilis R in the photo-steady state resulting from the red light
were collected offthe coast ofsouthern California during a exposure was estimated by comparing the integrated absor-
cruise ofthe RVNew Horizon between 12 May and 26 May bances of the two templates to the red light. This fraction
1996 using similar equipment and methods (see Kent, was then subtracted from the R/M mixture in the photo-
1997). Eyes were removed and cryopreserved for subse- steady state and a new template fitted to provide a better
quent microspectrophotometry, which was carried out at the estimate of the Amax of the M pigment. This traditional
University of Bristol, UK. Eyes were orientated in plastic analytical method assumes that all the visual pigment in the
wells filled with cryomount (Tissue Tek, OCT Compound, initial measurements made from the rhabdom was in the
Miles Inc., USA) and rapidly frozen with fluorocarbon form of R. The use of iterative template fitting methods
spray (Cryospray 22, Bright Instrument Co., UK). Frozen developed by Kent (1997) attempted to avoid the need for
blocks were individually sealed in plastic bags to avoid this assumption, and providedestimates ofthe proportion of
desiccation, placed in light-tight aluminum tubes, and main- M in the initial measurements and the Amax values of R
tained at 70C until sectioned for microspectrophotom- and M.
etry.
Electroretinogram experiments
Microspectrophotometry (MSP) experiments
Specimens used for electrophysiological recordings were
Frozen eyes from adult Sergestes siniilis were sectioned mounted underdim red light in a holder and suspended in a
using a cryostat and cut sections (average thickness 14 /urn) chamber filled with chilled (10C) seawater. This arrange-
were transferred to a 22 X 50 mm No. 1 coverslip, mounted ment allowed for enough pleopod movement to maintain
mm
in Tropic Marin artificial seawater, covered with a 19 respiratory currents across the gills. The electroretinogram
diameter No. 1 coverslip, and sealed with a ring ofsilicone (ERG), which is the summed mass responses from a large
grease. Sectioning proceeded from the region ofthe cornea number of photoreceptor cells, was recorded with a 10 /itm
most distal to the eyestalk towards the center of the eye. tip metal microelectrode (F. Haer & Co.) placed subcorne-
Visual pigment absorption was measured with a single ally underdim red light. The reference electrode was placed
beam, wavelength scanning, computer-controlled micro- in the other eye, which was covered with black Vaseline to
spectrophotometer described by Hart et <//., (1998). For block out light, and a silver-chloride electrode grounded the
invertebrate MSP, the instrument was modified by the in- water bath. Signals were amplified with an Xcell-3 Micro-
corporation ofa high intensity substage lamp that was used electrode amplifier (F. Haer & Co.), equipped with a high
for photoconversion ofthe visual pigment in the rhodopsin impedance probe to eliminate electrode polarization prob-
(R) state to the metarhodopsin (M) state using red light, and lems. Low frequency filters were set to minimal filtering
for the photobleaching of R and M mixtures to photoprod- (0.01-1 Hz) to minimize distortion due to AC amplification.
ucts of non-physiological importance using actinic white Monochromatic test flashes were provided by a tungsten
light exposure. This protocol is fully described by Kent light source, coupled to a grating monochromator (Instru-
mm
(1997) and follows the methods of Cronin and Goldsmith ments SA) with 1 slits in place, and delivered tothe eye
(1982). through one branch ofa bifurcated light pipe. The light pipe
Initial "baseline" scans were first made from 350 to 750 was placed 3 mm away fromthe eye, providing illumination
nm at 1-nm intervals in a tissue-free area of the cell prep- which covered the whole eye. Flash duration of 100 ms was
aration and then followed by several "sample" scans from controlled by a Uniblitz shutter (Model VS14S) undercom-
rhabdomeric tissue. Sample scans were first made from an puter control using LabView software (National Instru-
unexposed rhabdom which was then exposed to red light ments, Inc.). Irradiance was controlled with a neutral den-
(wavelength ofcut-on ca. 610nm) forapproximately 20sto sity wheel under computer control, calibrated in units of
photoconvert the rhodopsin pigment to a stable R/M mix- photons cm"2 *"1 with a UDT Optometer (Model S370)
ture, after which the rhabdom was re-scanned. The tissue and a radiometric probe placed 3 mm from the tip of the
was then photobleached by an exposure of approximately light guide. The adapting light source for chromatic adap-
30 min to white light and re-measured. Difference spectra tation experiments was an incandescent light filtered with a
between these data sets were calculated, from which the 400 nm or 480 nm broadband filter (Melles Griot). The
M
absorption spectra ofthe and R pigments in the rhabdom adapting light was delivered to the eye through the other
were determined by the method of Cronin and Goldsmith branch of the bifurcated light guide, ensuring that both the
(1982) as further developed by Kent ( 1997). In brief, visual adapting and test lights were acting on the same group of
pigment templates (Stavenga ct /., 1993; Palacios ct ul.. photoreceptorcells. Data were instantaneously analyzed for
VISION AND BIOLUMINESCENCE IN SERGESTES SIM/US 351
peak to peak response height using a program written in stimulus light chopper by means of a Scitec Instruments
LabView, digitized and stored to disk for later analysis. synchronizer. Thus, the photomultiplier measured only the
The eye was stimulated with 100 ms test flashes of bioluminescence produced by the test specimen and not the
monochromatic light adjusted for irradiance until either a stimulus light. The photomultiplier signal was processed by
100 or 200 fiV criterion response was obtained at each a Pacific Instruments amplifier/discriminator; square wave
wavelength tested. To ensure that the eye remained in the pulses were sent to a Newport Instruments model P6000A
same state of dark adaptation during the experiment, the frequency to voltage converter. The resulting voltage was
response to a flash of standard wavelength and irradiance measured using a Data Translation model 2801 data acqui-
was tested periodically throughout the experiment. Spectral sition board mounted in a personal computer. Data acqui-
sensitivity curves were generatedbased on the inverse ofthe sition, real-time display, and storage were controlled by a
irradiance required to produce the criterion response at each data collection program programmed using Data Transla-
wavelength. Absorptance spectra were constructed from tion DTVEE software. Bioluminescence and photodiode
visual pigment templates (Stavenga el al.. 1993). levels were continuously acquired at 2 Hz. Voltages were
subsequently converted to photon values based on a photo-
Behavioral sensitivity experiments metric calibration of the photomultiplier using 1 ml of
Cyalume* chemiluminescent liquid and a calibrated Quan-
Because Sergestessimilis produces bioluminescence only talum 2000 photometer. Bioluminescence in units of pho-
in the presence of a light stimulus, a chopped light source tons m " s was obtained by dividing photon flux (pho-
was used to avoid detection ofthe stimulus illumination by tons s~') by thecross-sectional areaofthe ventral surface of
mm
the light detector (see below). Bioluminescence was mea- the organs of Pesta for an adult shrimp of size 14
sured in the briefdark intervals when the stimulus was off, carapace length (Latz. 1983).
similar to the methods ofWarner el al. ( 1979) and Latz and To measure stimulus intensity, a Graseby Optronics
Case (1992). Illumination from a Dolan-Jenner model 180 model 260 calibrated sensor head was mounted in the inte-
tungsten-halogen source was conducted through an optical grating sphere in the same position as the specimen cham-
fiber bundle to an Optometrics model DMC1 monocbroma- ber. Light levels measured in watts with a Graseby Optron-
tor and then through another optical fiber bundle to a JML ics model S370 Optometer were converted to irradiance
Optical electro-mechanical shutterthatcontrolled the timing units of photons m"2 s~'.
ofthe "on" and "off transitions of the stimulus. The stim- Quantum sensitivity. To determine the threshold level of
ulus light was chopped at 80 Hz using an Oriel variable light that prompts luminescent countershading by Sergestes
frequency chopper, attenuated by neutral density filters to similis, bioluminescence produced by induced animals in
control light intensity, and diffused by a single layer of response to various intensities of light was measured. To
glassine paperbefore entering the test chamber. Because the induce luminescent countershading. dark-adapted animals
critical flicker-fusion rate at such low irradiances is <60 Hz were exposed to a standard light stimulus (490 nm. 2.24 X
for marine crustaceans (Waterman. 1961; Frank. 1999), the 10" photons irT2 s"1) for 25 mm (Latz and Case. 1992);
test animal perceived the chopped stimulus light as contin- wavelength and irradiance settings were chosen based on
uous. preliminary experiments. After induction, shrimp were ex-
For testing, specimens were loosely restrained by a clear posed to a series of test stimuli as follows: 60 s darkness,
acrylic clamp around the cephalothorax and placed in a 60 s test stimulus, 60 s darBkness. 5 or 10 min standard
sealed, clear acrylic chamber (1.75 X 2.5 X 10 cm) filled stimulus (490 nm, 2.24 x 10 photons m~2 s~'), repeating
with 10-1 1C seawater. The clamp allowed free movement this pattern with new test stimuli until all irradiances were
of pleopods and other appendages, yet prevented the spec- tested, typically within 2 h. The purpose of the standard
imen from shifting position. Throughout an experiment, stimulus was to maintain shrimp in the light-adapted in-
seawater chilled to 1 1-12C by a Fisher Scientific model duced state for luminescent countershading so that their
1016S recirculating chiller was recirculated through the bioluminescence responses to the test stimuli would show
chamber at a rate of 100 ml min~' using a Masterflex fast kinetics representative of luminescent countershading
peristaltic pump. The specimen chamber was placed in the (Latz and Case. 1992). In addition, the responses to the
center ofthe light collection chamber, which consisted ofa standard stimulus were used to monitorthe condition ofthe
25 cm diameter Labsphere integrating sphere. The advan- animal, and. if necessary, correct for changes in animals"
tage of an integrating chamber is that the measurement of responsiveness over time (see data correction below). The
emitted light is minimally affected by photophore oranimal duration of the standard stimulus was determined by the
position. Bioluminescence was detected by a Burle model return to the level ofbioluminescence measured during the
8850 photon-counting photomultiplier operating at 1790 initial induction. Typically, this occurred within 5 min, but
V, after passing through a second optical chopper operating occasionally, after the dimmest irradiance test stimuli. 10
at 80 Hz but synchronized 180 out of phase with the min of the standard stimulus was required. Test stimuli of
352 S. M. LINDSAY ET AL.
490nmlight were used at the following irradiance levels (in cence intensity and stimulus intensity holds at all levels of
units of photons irT2 s~'): 2.22 >: 10", 3.45 x 10", light adaptation or for changes in specimen responsiveness
6.41 X 10", 3.95 X 1012, 1.01 X 1013, 2.24 X 1013. 3.40 x due to fatigue. This is an important assumption because in
1013, 3.87 x 1013, 4.98 x 1013. 7.65 X 1013, 9.72 X 1013. 4 of 9 spectral efficiency experiments, bioluminescence
Spectral sensitivity: To measure the spectral efficiency of intensity showed slight but significant decreases over the
luminescent countershading. dark-adapted animals were course of the experiment. Nevertheless, in all cases the
first exposed to a 490-nm stimulus (at 2.24 X 1013 photons bioluminescent responses to test stimuli showed the char-
m~2 s~') for25 minto inducecounterillumination. Animals acteristic fast kinetics indicative ofthe induced counterillu-
were thenexposedto light stimuli at 20-nm increments from mination condition (Latz and Case, 1992). In those 4 spec-
400 to 640 nm. following the general protocol described imens demonstrating a decrease in the standard response at
above: 60 s darkness, 60 s test wavelength stimulus, 60 s 490 nm. a correction factor for each data point for each of
darkness, 5 or 10 min ofthe standard illumination to main- these individuals was determined based on a linear regres-
tain the induced countershading state. This process was sion describing the intensity ofbioluminescence in response
repeated for each wavelength tested. The average duration to the standard stimuli as a function of time.
of a complete trial was 3 h. The order of test wavelengths The effects of these corrections are shown for a single
was randomized. Because shrimp eyes operate as photon individual (Fig. 1). Responses were corrected for a standard
counters, and the total number of photons in a given stim- stimulus irradiance of 1.20 X 1013 photons m~2 s~' as
ulus is a function of both light intensity and wavelength, shown in the following example. For this individual, the
stimulus irradiance was adjusted at each wavelength using relationship between bioluminescence irradiance (in origi-
neutral density filters to obtain equal photon irradiance nal unitsofvolts s~') and stimulus irradiance at490nm (the
levels at each wavelength. Even so, there were slight vari- standard stimulus wavelength) is best described by the
ap1th.io1o6tnosnXsin1i0rsT1t32impsuh~lo'utsoantisr6rn2a0dTia2nnmsc.~e'Blaeitvoel4lus0,m0inwnehmsiccethoncr2ea.n5d2gaetdXa fw1re0ro1em3 er(rqe-ugar=teiso0sn.i4:o0n,bieFoqlu=uatmi2io0nn.e,0s3c,tehnde.cfe.pr==edi31c..3t61e6,dXPbi=1o0l~0u.hm0i0*n0e1isr)cr.aedUniscaienncgeatt'3hains
dbssatuiirbomdslueulqmiuuirsern,naedtsailcanyendnccceoterhorewfeaclsta1es.bdt2a0sa4seXdmdeiotnn1a0itlfheoedrplhabtsohettel2oo0nswstsatnomodfareerafdlcehc5t-601a-0Ms-setmtaaeinsnn-t siirrrr1aa.ddFiioaannrccaeetwoefasts1w.1a2.v02e8lXeXn1g01t01h313opfphh4oot0to0onnnssmm,m"t"h2e2sms"e1a1s.isuTrh0e.ed48psr0te5idmivucoltluetssd
bioluminescence (using the regression equation) for a 490
illumination.
nm stimulus at this irradiance level is 0.4924 volts s"',
Data correction. Because the test stimuli in the spectral
ewseaencrsheitwcivoairvtreyelceetxnepgdetrthoimbreaefnslteesdctvotanhreiaeidnsttseilnmisugilhttuylsyofiirnbriaidoniltaeunnmcsieinteyo,sfcae1lln.2cd0eataXat gf1oi0rv1i3ang4ph9ao0t-coonnrmsrescmtti~imou2nlsuf~sa',ctbiosiro0lo.uf9m70i6.n9et7si6cmee(ns0c.et4h8ia0nt5t/ean0ts.i41t9.y224a8)t.X1.T2h10u0sX1,3
10 photons m"2 s~'. Data collected in the quantum sen- photons m~2 s"1. Making the important assumption that
bsiettivwieteynebxipoelruimmiennetsscewnecreeinutsenesditytoanmdodsetilmutlhues icnotrernesliattyioant qleunagntht,umthesenbsiiotlivuimtiynedsoceesncneotvaclhuaengmeeawsiutrhedstiatmu4l0us0 wnamvei-s
490 nm. One of two regression equations (for either San multiplied by 0.976 to reflect the response to a "standard"
Diego or Santa Barbara collected specimens) was used to
calculate (1) the predicted bioluminescence at 1.20 X 1013
photons m~2 s~', and (2) the predicted bioluminescence at " 20-,
the irradiance level measured foreach test wavelength stim- E 18-1
ulus. Dividing (1) by (2) gave a proportional correction <6-
factor that was multiplied by the bioluminescence value at 1d 14-
12-
each test wavelength. Because not enough specimens were
available to empirically derive the relationship between
stimulus intensity and bioluminescence intensity at every
wavelength tested, the assumption was made that quantum
sensitivity did not change with stimulus wavelength, so that
the relationship observed at 490 nm holds for the other 380 420 4W6a0ve5l0e0ngt54h0(n5m8)0 620 660
wavelengths. This assumption of univariance is supported
by the visual sensitivity and MSP experiments which dem- olFSiig'ruxreexte1.x xiCmoirlrise.ctUinoncoorfrescpteectdradlateaffi(csioleindcycidractlaesf)orwaerseingcloerrsepcteecdimfeonr
onstrated that only a single visual pigment is present. variations in stimulus intensity (opencircles) and forboth stimulus inten-
The correction for variable stimulus intensity also as- sity and temporal decrease in responsiveness (closed triangles). Reter to
sumes that the observed relationship between biolumines- Materials and Methods fordetails on data correction.
VISION AND BIOLUMINESCENCE IN SERGESTES SIMILIS 353
stimulus of 1.20 X 1013 photons m 2 s '. This correction 0.2-i
was made in turn for each stimulus wavelength.
0.15-
Data from this individual were also corrected for a de-
crease in responsiveness, because the least-squares linear
0.1-
regression between bioluminescence intensity (in volts s )
and the order of presentation of the 490 nm standard stim- 1 0.05-
ulus showed a slight but significant decay in response ac-
=
cording to the following equation: bioluminescence
-0.022 * (order of presentation) + 0.887 (r = 0.72, F =
36.35, d.f. = 1,15, P < 0.0001). Because the response to -0.05300 400 500 600 700
490 nm standard stimuli was somewhat variable over time, Wavelength(nm)
thisregressionequationis thebestdescription ofthe general
decay in response. The decay correction factor was calcu-
lated based on time of stimulus presentation by first calcu-
lating the predicted bioluminescence at a given stimulus
time using the previous equation, and then dividing that
result by the bioluminescence measured at the first 490 nm I 0.6
standard stimulus. For example, bioluminescence in re-
sponse to the fifth 490 nm standard stimulus was calculated 0.2-
to be 0.7771 using this regression equation. Dividing this
value by the value for the first standard stimulus (0.7857)
-0.2
gives a decay correction factor of 0.989. Making similar 300 400 500 600 700
calculations for the order ofpresentation ofeach test wave- Wavelength(nm)
length stimulus, the bioluminescence value for each test Figure 2. Spectral absorbance based on microspectrophotometry of
stimulus was divided by the appropriate decay correction fivesectionsofrhabdomerictissuefromtheretinaofSergesressimilis. (A)
factor. The decay correction assumes that the effects were Averagedabsorbancespectra;boldtraceshowstheinitialabsorbance,light
equivalent at all wavelengths tested. Following corrections trace shows the absorbance following saturating red light illumination,
for stimulus intensity and response decay, all biolumines- dashed trace shows the absorbance following photobleaching with bright
cence data were converted from units of volts s~' to pho- white light. Fordisplay, spectra have been standardized to an absorbance
tons m~2 s"1 as previously described, based on the photo- ospfezcetrroa aftor73p0hontmo.bltehaechliimnigtoofftthheesrpehcatbradlomscafnr.om(B)itsAvienirtaiagledstdaiteffe[rbeonlcde;
metric calibration of the photomultiplier and the cross- derivedfromboldtraceminusdottedtracein(Al]andforthephotobleach-
sectional area of the ventral surface of the organs of Pesta ingofthe rhabdomfromitssteady state R/M mixturefollowingsaturating
for an adult shrimp of size 14 mm carapace length (Latz, redlight[light;derivedfromlighttraceminusdottedtracein(A)].Specific
1983). To directly compare visual and behavioral spectral aabnsdo0r.ba0n0c9e4s/Mat,m~t'h,erAemsapxectfiovreltyh.esSemaobostohrbsaonlciedtsrpaeccetsraarweerbeest0-f.i0t0t7e8mpjluamt~e's
sensitivity, irradiance values from the electrophysiological (Stavenga etal.. 1993) with Amax values of492 nm and 485 nm. respec-
eioxrpaelreixmpeenrtismeanntdsbwieorleumnionremsacleinzceedvfaolrueesacfhrionmditvhieduable.hav- ttirvuemly.(ATmhaex =das4h8e4dnlmi)neriessutlhteinegstfirmoamtetdhemecotrarrehcotdioonpsfionrathbesorrebsaidnucaelsMpeci-n
the R/M mixture difference spectrum (light trace), and the absorbance
Results spectrum oftheMrhodopsin (dotted; Amil, = 495 nm), aftercorrecting for
contaminating in the initial scan (bold).
Microspectrophotometry (MSP)
Spectral absorbance,basedon MSP measurements offive initial measurements may have been as high as 15% and,
sections ofrhabdomeric tissue from a single individual, was after correction for this contamination, the R and M Amax
unimodal with maximum absorbance in the blue-green (Fig. values can be revised to 495 nm and 484 nm, respectively,
2A). Assuming that the initial scans were uncontaminated with a M/R extinction ratio of 1.406.
with metarhodopsin (M) pigment, template fitting to the
difference spectra (Fig. 2B) indicated a rhodopsin (R) pig- Visual sensitivity ofthe eye (ERG)
ment with a Amax of 492 nm. The template best-fitting the
R/M mixture after red light exposure yields a Amax of 485 The electrophysiologically determined visual spectral
nm. This value was corrected to allow for the residue of R sensitivity of dark-adapted specimens of Sergestes similis
in the R/M mixture to give abest estimate ofthe M pigment indicated that the sensitivity maximum was centered at
Amax of484 nm, and an M/R extinction ratio (at the respec- approximately 500 nm in the blue-green region ofthe spec-
tive Amax values)of 1.333. Iterativetemplate-fitting methods trum (Fig. 3). Both blue (480 nm) and near-UV (400 nm)
M
(Kent, 1997) suggest, however, that the fraction of in the chromatic adaptation uniformly depressed the sensitivity
354 S. M. LINDSAY ET AL.
-0.6-1
0.8-
5 0.6-
0.2-
380 420 460 500 540 580 620
Wavelength(nm)
Figure 3. Visual spectral sensitivity of Sergestes similis based on
electroretinogram (ERG) measurements. Sensitivity, based on the inverse
of the irradiance required to elicit a !()() or 200 /u.V response, showed a
broad maximumcenteredaround500nm. Symbolsrepresentmeans SE
for6 specimens.
curve across the spectrum (Fig. 4), and had no effect on
ERG waveform (Fig. 5). These results are consistent with
MSP
those of the study indicating that only a single visual
pigment is present.
Behavioral sensitivity
The magnitude of luminescent countershading by 5. si-
milis depended on the level of stimulus irradiance (Fig. 6).
-8.0-,
darkadapted
480nmadapted
-9.0-
y>
0-10.0-
o
jz
Q.
roa-11.0-
-12.0
380 420 460 500 540 580 620
-8.0-, B
darkadapted
400nmadapted
-9.0-
\
0-10.0-
VISION AND BIOLUMINESCENCE IN SERGESTES SIM1L1S 355
v, 4.5 6.4x1011 3.9x1012 10x1013 2.2x101
photonsm s"
50
Figure6. Representative bioluminescent responses by a single specimen ofSergestessimilis to increasing
stimulus irradiance. Bioluminescence was first induced using a490nm stimulusofintermediate intensity, then
the specimen waspresented with 60-s stimulifrom a rangeofintensities, all at490nm.The linerepresentsthe
levelofbioluminescenceversustime.Darkbarsindicatestimuluslightoff;whitebarsindicatestimuluslighton.
Values above graphs are stimulus intensities.
mm
cated that S. similis possesses a single visual pigment with experiments, which ranged in size from 10.3 to 13.5
maximum absorbance around 495 nm. Such data can be carapace length, the estimated upper and lower bounds for
used to calculate the spectral sensitivity of a photoreceptor rhabdom lengths in these animals were approximately 120
containing this visual pigment by first calculating the spec- to 150 /xm, with a mean of approximately 135 ju,m. This
tral absorptance of an axially illuminated rhabdom. To do value is similar to that reported by Hiller-Adams et al.
this, two additional pieces of information are required: (1988) for the sergestid Sergia tenuiremis. MSP measure-
rhabdom length and the specific absorbance (i.e.. absor- ments ofspecific absorbance forS. similis suggest aspecific
bance jumT1) of the visual pigment in the photoreceptor. absorbance of 0.0074 /uirT1 (Kent, 1997) although this is
Rhabdom lengths vary somewhat with eye size, but by significantly lower that the value of0.01 jam"1 reported by
sectioning the aldehyde preserved eyes of 5. similis, rhab- Cronin and Frank (1996) in Systellaspis debilis, but only
dom lengths were found to range from 128 to 161 /urn for slightly lower than the value of 0.008 ju.m~' reported as
mm
shrimpswithcarapace lengthsof 1 1.8 to 14.2 (T. Frank, being 'typical' of crustacean photoreceptors (e.g., Cronin
ERG
unpublished data). For the specimens used in the and Goldsmith, 1982). At wavelengths greaterthan the peak
absorbance (495 nm). the spectral absorptance closely
matches spectral sensitivity data from ERG measurements
1X101 and the spectral efficiency curve of luminescent counter-
shading (Fig. 10). Using maximum or minimum values
instead ofmean rhabdom length has little effect on spectral
absorptance, while increasing specific absorbance to levels
1X101 more typical of crustacean photoreceptors leads to a better
fit between the different data sets at long wavelengths. At
short wavelengths, however, there is significant divergence
which probably cannot be attributed to the photosensitivity
1x101 spectrum of the rhodopsin departing from the absorptance
spectrum at short wavelengths, although dataonthis subject
5 1x10"
5 1x10i3 are limited (Dartnall, 1972). It is more likely that the effec-
1x10 1x1012 1x101 1X101 tive spectral sensitivity ofthe eye is affected by intraocular,
Irradiance(photonsm'Js ) pre-retinal filters which selectively filter short wavelength
cenFcieguprreod7u.cedEfbfyectSeorfgelsitgehst siinmtielnissi.tyAlolnsativmeurlaigewemraexaitmaumwavbeiloelunmgitnheso-f ligEhlte(cGtorlodpshmysiitohl,og1i9c7a8l;mreeavsiuerweedmebnytsFeuisninagndthSezuetlse,ctr1o9r8e2t)i.-
490 nm. The magnitude ofbioluminescence measuredduringthelast 20s nogram (ERG), corresponding to the summed mass re-
ofeach60-steststimuluswasaveragedforeachindividual.Tenspecimens sponse of a large number of photoreceptor cells to a light
weretested; means SEare shown. Responsesforstimulus irradiance > stimulus, were performed to determine the behaviorally
e2vaqluXuaets1i,0on12liypghh=totlo(en1vs.e8lm7s~Xw2ersl~eO'^tnwxee"arr1e5b(baercskt=gdre0os.uc6nr1d)i..beAdNtobtyleotwhteehrepsostweipemarurlalutasewisr(crlaaoldgei-alnofcgoe)r rneilqeuveanptrosvpiecdtersalasemnosriteivcitoymporfehS.en.ssiimvileis.asWsheislsmeentthisoftecthh-e
above-threshold bioluminescence. visual spectral sensitivity of an organism than do measure-
356 S. M. LINDSAY ET AL.
420nm 500nm 580nm 600nm
50
Figure 8. Representative bioluminescent responses by a single specimen ofSergestes similis to different
wavelengthsoflight. Bioluminescencewasfirstinducedusinga490nmstimulusofintermediateintensity,then
thespecimenwaspresentedwith60sstimuliatvariouswavelengths,atapproximatelyequal irradianceof1.5 x
10'-' photons m~-s '. Bars as for Figure 5.
ments from single photoreceptors (reviewed by Goldsmith. ulus and response as well as calibration assumptions make
1986). ERG results do not reflect the amount ofhigherorder direct comparisons difficult, although relative changes
processing of visual input, nor the behavioral response to should still be valid. In the present study, the range of
visual stimuli. Thus the behavioral studies of luminescent stimulus irradiance tested was less than two orders of mag-
countershading extend the physiological assessment, result- nitude. Within this range, bioluminescence increased with
ing in a comprehensive description of the organism's sen- stimulus irradiance according to a power law (log-log)
sory and behavioral response to ecologically relevant light regression as found by Young et al. (1980) for midwater
stimulation. squid and fish. However, the magnitude of the increase in
Luminescentcountershading by S. xiniilisoccurredovera bioluminescence did not match the magnitude of the in-
relatively narrow range of irradiance. A behavioral thresh- crease in stimulus irradiance. Other counterilluminating an-
oldoccurredatapproximately 3 X 10|: photons m~2 s~', as imals may not precisely match changes in the stimulus
lower irradiance levels resulted in minimal levels of biolu- irradiance (Young et al., 1980). yet they exhibit a better
minescence which were not significantly different from match over a larger dynamic illumination range for lumi-
background. This illumination level may represent the min- nescent countershading than did 5. similis in the present
imum irradiance causing light adaptation of the eye. which study.
appears tobe required forluminescent countershading (Latz In Sergestes similis, behavioral spectral efficiency was
and Case. 1992). Under ideal conditions, bioluminescence similar to visual spectral sensitivity. A survey ofspecies for
should exactly match stimulus irradiance. As discussed by which behavioral and physiological spectral sensitivity data
Young et al. (1980), differences in geometry between stim- are available (Table I) suggests that behavioral spectral
I ,,H
" 09-
380 420 460 500 540 580 620 660
3 380 420 460 500 540 580 620 660 Wavelength(nm)
Wavelength(nm)
Figure 10. Comparisonofrelativevisual and behavioral spectral sen-
Figure9. Behavioral spectral sensitivityofSergestessimilis basedon sitivity of Sergesres similis. Symbols represent means SE. Both the
thebioluminescencespectral efficiency,correctedforastimulusirradiance normalized ERG sensitivity (solid circles) and normalized biolumines-
of 1.2 X 10" photonsm~~ s"1 (seetext). Symbols representmeans SE cence spectral efficiency (open circles) coincide well at long wavelengths
for9 specimens;the curve is a fourthdegreepolynomial function fitted to with the calculated spectral absorbance (solid line) of a rhodopsin with
thedata, wherey = 6.1 X 10V - 1.2 X 10V + 9.2 x 10'V - 3.0 x peak absorbance at 495 nm. a rhabdom axial length of 135 /urn and .1
10"x + 3.6 x 1015. specific absorbance of0.0074 /xn-T1.
VISION AND BIOLUMINESCENCE IN SERGESTES SIMII.IS 357
